Abstract
Aminoglycoside 6’-acetyltransferase-Im (AAC(6’)-Im) is the closest monofunctional homolog of the AAC(6’)-Ie acetyltransferase of the bifunctional enzyme AAC(6’)-Ie/APH(2”)-Ia. The AAC(6’)-Im acetyltransferase confers 4- to 64-fold higher MICs to 4,6-disubstituted aminoglycosides and the 4,5-disubstituted aminoglycoside neomycin than AAC(6’)-Ie, yet unlike AAC(6’)-Ie, the AAC(6’)-Im enzyme does not confer resistance to the atypical aminoglycoside fortimicin. The structure of the kanamycin A complex of AAC(6’)-Im shows that the substrate binds in a shallow positively-charged pocket, with the N6’ amino group positioned appropriately for an efficient nucleophilic attack on an acetyl-CoA cofactor. The AAC(6’)-Ie enzyme binds kanamycin A in a sufficiently different manner to position the N6’ group less efficiently, thereby reducing the activity of this enzyme towards the 4,6-disubstituted aminoglycosides. Conversely, docking studies with fortimicin in both acetyltransferases suggest that the atypical aminoglycoside might bind less productively in AAC(6’)-Im, thus explaining the lack of resistance to this molecule.
Highlights
Aminoglycosides are potent, broad-spectrum bactericidal antibiotics used to treat many serious bacterial infections [1]
The major mechanism of aminoglycoside resistance in Gram-positive pathogens is the production of aminoglycoside-modifying enzymes, which include aminoglycoside acetyltransferases (AACs), aminoglycoside phosphotransferases (APHs), and aminoglycoside nucleotidyltransferases (ANTs) [5,6,7,8]
Antibiotic resistance profile When expressed in the Escherichia coli JM83 strain, AAC(6’)-Im produces resistance to a wide range of clinically important 4,6-disubstituted aminoglycosides with minimal inhibitory concentration (MIC) values 8 - 128-fold above those for the recipient strain (Table 1)
Summary
Aminoglycosides are potent, broad-spectrum bactericidal antibiotics used to treat many serious bacterial infections [1]. We report the antibiotic susceptibility and kinetic profile and the X-ray structure of the AAC(6’)-Im acetyltransferase in its apo form and as the binary kanamycin A complex, and classify the enzyme as a member of the AAC(6’)-I sub-family C.
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